Research news

For the first time, scientists have described in this week's Science a way for cells to add phosphate groups to proteins that doesn't involve using adenosine
triphosphate (ATP) as a donor (Science 2004, 306:2101-2105).

"Nobody had ever dreamt you could phosphorylate with a donor other than ATP," said
senior author Solomon Snyder at Johns Hopkins University in Baltimore, who, along with colleagues, suggested a decade ago that inositol pyrophosphates such as diphosphoinositol pentakisphosphate
(IP7) might serve as phosphorylating agents due to their highly energetic pyrophosphate
bonds.

"ATP phosphorylation has heretofore been regarded as the primary mode of all cellular
signaling in biology," Snyder told us. "IP7 phosphorylation may be of comparable importance,
with similarities but major differences - for instance, it is nonenzymatic. It may
represent a new form of intracellular signaling."

In the current study, the group synthesized IP7 and radiolabeled the putative donor
pyrophosphate. As a control to ensure any apparent phosphorylation did not simply
reflect binding of IP7 to proteins, the researchers also created IP7 with a different
radiolabeled phosphate group.

In mouse brain and yeast extracts, gel electrophoresis and autoradiography revealed
the radiolabeled IP7 phosphorylated as many proteins as radiolabeled ATP, while the
control did not. "IP7 looks to act quite universally," Snyder said. "And while the
numbers are similar, it looks as if IP7 and ATP are phosphorylating different proteins."

"What's also remarkable is we have evidence not just of phosphorylation of proteins,
but of pyrophosphorylation, adding a phosphate on top of a phosphate. That's completely
unheard of," Snyder said.

IP7 phosphorylation appears selective for eukaryotic organisms, with none seen in
Escherichia coli extracts. Phosphorylation patterns also differed among mouse brain, mouse kidney,
and Drosophila melanogaster. IP7 requires magnesium as a cofactor just as enzymes for ATP do.

The primary objective now, said Stephen Shears of the National Institute of Environmental Health Sciences in Research Triangle Park,
NC, is to determine whether IP7 phosphorylation has any biological significance in
vivo.

"It's not going to be easy," said Shears, who did not participate in the study. "Experiments
that approach the problem by changing levels of IP7 in intact cells will never be
specific enough, because so many additional off-target effects will result; the inositol
pyrophosphates are pleiotropic."

"One very direct approach: Identify an IP7 target protein with a defined function
that is assayable with intact cells. Then, identify an amino-acid residue on that
target that is only phosphorylated by IP7 and not by other protein kinases. Several
candidate IP7 targets may need to be screened," Shears told us. "Having done that,
express in cells a single site mutant version of this protein that cannot be phosphorylated
by IP7. This will be a kind of 'dominant negative.' Now, perform bioassays that are
normally done for the protein in question and see if its function is impaired."

John York of Duke University in Durham, NC, who co-wrote a commentary on Snyder's work, said
future research should address what the specificity and stereochemistry of IP7 binding
is, how phosphate transfer is activated, and how a target serine or phosphoserine
is selected.

"It's puzzling that if you produce yeast NSR1 in bacteria as a recombinant protein,
the transfer of phosphate does not occur. That suggests a priming step done by the
yeast cell prior to transfer, which is a little peculiar, and requires a follow-up
study to characterize," York told us.